1. Field Of The Invention
This invention relates to a concentration detector device containing a set of electrodes and a composite membrane structure wherein an enzyme, specific to a substance found in a fluid sample, is immobilized. The concentration of the substance found in the fluid sample is determined by proportional comparison with the amount of current generated from the oxidation of the product of an enzymatic reaction at the anode of the detector device. This invention also relates to methods for determining the concentration of glucose or other substrates in blood, body fluids and other solutions using this detector device.
2. Description Of The Prior Art
For medical and research purposes it is often important to accurately and rapidly measure the concentration of certain substances found in body fluids, such as glucose. For example, it is critically important to rapidly and accurately monitor blood glucose levels in diabetic patients who require injections or infusions of appropriate dosage levels of insulin to control blood glucose levels. Additionally, blood glucose determinations can be important when a diabetic patient is acutely ill, undergoing surgery or childbirth or suffering from severe keto-acidosis.
The prior art describes a number of detector devices for electrolytically analyzing the concentration of a substrate by measuring electrically the components produced by reaction of an enzyme specific to a substrate contained in a sample.
For example, Clark U.S. Pat. No. 3,539,455 describes a device and system wherein glucose, in a body fluid sample, is oxidized by glucose oxidase to convert the same to gluconic acid and hydrogen peroxide, the latter being detected polarographically. Other interfering materials in the sample which result in extraneous currents according to Clark are compensated for by current subtraction.
Newman, U.S. Pat. No. 3,979,274 (1976) discloses a system where a membrane laminate contains glucose oxidase enzyme in an adhesive layer in close proximity to a reference electrode.
Wilkins, U.S. Pat. No. 4,440,175 (1984) describes a implantable electrode which includes a membrane formed on a conductive substrate, the membrane containing a polymeric anion exchange species with a water soluble salt of the nonionic species to be measured. The device is used to measure glucose concentration in vivo.
Shichiri et al., "Wearable Artificial Endocrine Pancreas With Needle Type Glucose Sensor", Lancet, Nov. 20, 1982, pp. 1129-31, describes an implantable needle electrode for monitoring glucose which has at the end a urethane ball structure containing immobilized glucose oxidase (in cellulose-diacetate with a heparin anticlotting agent) surrounding a platinum electrode (anode) and a silver cathode "body" surrounding the anode and separated therefrom by a glass wall or tube.
The present detector device incorporates several design features which permit quick determinations of substrate concentration more accurately and reliably than the prior art. Particularly, the detector device of the present invention can be used to accurately and reliably measure the concentration of glucose in solution.
To measure glucose concentration the enzyme glucose oxidase is immobilized within a detector cell near a set of planar electrodes. A solution containing glucose is deposited in the detector device cell. The immobilized enzyme, glucose oxidase (GO) causes the catalysis of glucose to gluconic acid and hydrogen peroxide in accordance with the general Reaction (1), below: ##STR1##
This reaction is described in Reed, G. Enzymes in Food Processing, Academic Press, NY (1975), p. 231 as follows: ##STR2##
The enzyme glucose oxidase (termed E-FAD in the above reaction) is said to extract two hydrogens from .beta.-D-glucose to form the reduced enzyme, EFADH.sub.2 and the gluconic acid lactone. The lactone hydrolyzes in the presence of water to gluconic acid (nonenzymatically) and the reduced enzyme E-FADH.sub.2 is reoxidized by molecular oxygen to E-FAD.
The resulting hydrogen peroxide produced as shown in reaction 1-A is then oxidized at the Pt electrode held between +0.7 to +0.9 volts in order to produce electrons and current, as shown in Reaction (2) below: EQU H.sub.2 O.sub.2 .fwdarw.2H++O.sub.2 +2e- (2)
The current generated in Reaction (2) is thus directly proportional to the concentration of glucose found in the sample which is deposited in the detector device cell, provided there is sufficient oxygen to carry out the regeneration step in Reaction (1-A). A source of oxygen can also be provided to facilitate the regeneration of glucose oxidase according to Reaction (1-A).
The generating of available oxygen in the reaction volume present in the sample cup or reservoir by the electrolysis of water offers significant operational advantages to the detector system of this invention. It is noted that in situ generation of oxygen by the electrolysis of water is discussed by Enfors in Enzyme Microb Technol., 3, 29-32 (1981) with respect to investigative efforts to develop a regeneratable glucose oxidase electrode which could be used to continuously measure the concentration of glucose of a fermentation broth.